Instruction Manual Tag-On-Demand TM Suppressor Supernatant A recombinant adenovirus expressing a tRNA ser suppressor to facilitate transient expression of C-terminally-tagged proteins in mammalian cells Catalog nos. K400-01, K405-01, K410-01, K420-01 Version C October 31, 2010 25-0659 A Limited Use Label License covers this product (see Purchaser Notification). By use of this product, you accept the terms and conditions of the Limited Use Label License.
Transcript
Instruction Manual
Tag-On-DemandTM Suppressor SupernatantA recombinant adenovirus expressing a tRNAser suppressor to facilitate transient expression of C-terminally-tagged proteins in mammalian cells
Catalog nos. K400-01, K405-01, K410-01, K420-01
Version COctober 31, 201025-0659
A Limited Use Label License covers this product (see Purchaser Notification). Byuse of this product, you accept the terms and conditions of the Limited Use LabelLicense.
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Table of Contents
Table of Contents ............................................................................................................................................... iii Kit Contents and Storage ................................................................................................................................... v Accessory Products........................................................................................................................................... vii
Introduction ..................................................................................................................1 Overview...............................................................................................................................................................1 The Tag-On-Demand� System ..........................................................................................................................3 Biosafety Features of the Tag-On-Demand� Suppressor Supernatant ........................................................5 Experimental Outline ..........................................................................................................................................6
Methods ........................................................................................................................8 Storing and Handling the Tag-On-Demand� Suppressor Supernatant ......................................................8 Generating the Expression Construct ...............................................................................................................9 General Guidelines to Use the Tag-On-Demand� System ..........................................................................11 Using the Tag-On-Demand� Suppressor Supernatant ................................................................................15 Detecting GFP-Tagged p64TAG Protein............................................................................................................21 Examples of Expected Results..........................................................................................................................23 Troubleshooting.................................................................................................................................................26
Appendix.....................................................................................................................28 Map of pcDNA�6.2/GFP-GW/p64TAG ...........................................................................................................28 Invitrogen Vectors Compatible with the Tag-On-Demand� System .........................................................29 Technical Service................................................................................................................................................31 Purchaser Notification ......................................................................................................................................33 Product Qualification ........................................................................................................................................35 References ...........................................................................................................................................................36
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Kit Contents and Storage
Types of Kits This manual is supplied with the following products. Catalog numbers K410-01
and K420-01 are also supplied with a Tag-On-Demand� Gateway® Vector and the Tag-On-Demand� Gateway® Vectors manual. For more information about the Tag-On-Demand� Gateway® vectors, see below.
Kit Components The Tag-On-Demand� Gateway® Kits include the following components. For a
detailed description of the contents of each component, see the next page.
Note: Catalog nos. K400-01 and K405-01 include the Tag-On-Demand� Suppressor Supernatant and a vector control only.
Component Catalog no.
K400-01 & K405-01 K410-01 K420-01
Tag-On-Demand� Suppressor Supernatant √ √ √
pcDNA�6.2/V5-DEST Gateway® Vector √
pcDNA�6.2/GFP-DEST Gateway® Vector √
Shipping/Storage The Tag-On-Demand� products are shipped as described below. Upon receipt,
store each item as detailed below.
Box Shipping Storage
Tag-On-Demand� Suppressor Supernatant
Dry ice Suppressor Supernatant: -80°C
Control plasmid: -20°C
Tag-On-Demand� Gateway® Vector
Room temperature
-20°C
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Kit Contents and Storage, continued
Tag-On-Demand� Suppressor Supernatant
The following reagents are included with the Tag-On-Demand� Suppressor Supernatant. Store the Tag-On-Demand� Suppressor Supernatant at -80°C and the control plasmid at -20°C. Note: The pcDNA�6.2/GFP-GW/p64TAG control is not included with Catalog no. K420-01.
Reagent Composition Amount for K400-01,
K410-01, K420-01 Amount for
K405-01
Tag-On-Demand� Suppressor Supernatant
CsCl-purified, recombinant adenovirus supplied at a titer of 1 x 109 pfu/ml in:
10 mM Tris-HCl, pH 7.5
1 mM MgCl2
150 mM NaCl
10% glycerol
200 µl 5 x 200 µl
pcDNA�6.2/GFP-GW/p64TAG control plasmid
Lyophilized in TE, pH 8.0 1 µg 1 µg
Number of Transductions
The amount of Tag-On-Demand� Suppressor Supernatant supplied is sufficient to transduce mammalian cells at an MOI of 50 in the following formats:
Cat. nos. K400-01, K410-01, and K420-01 Cat. no. K405-01
• One complete 6-well plate or
• One complete 24-well plate or
• Two complete 96-well plates
• Five complete 6-well plates or
• Five complete 24-well plates or
• Ten complete 96-well plates
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Before using the Tag-On-Demand� Suppressor Supernatant, read the section entitled Biosafety Features of the Tag-On-Demand� Suppressor Supernatant, page 5 for safety information and the section entitled Storing and Handling the Tag-On-Demand� Suppressor Supernatant, page 8 for storage and handling information.
Tag-On-Demand� Gateway® Vectors
The Tag-On-Demand� Gateway® Kits (Catalog nos. K410-01 and K420-01) include a Gateway® destination vector (i.e. pcDNA�6.2/GFP-DEST or pcDNA�6.2/V5-DEST) for cloning your gene of interest and a corresponding expression control vector. Refer to the Tag-On-Demand� Gateway® Vectors manual for detailed information pertaining to each expression vector and a description of the reagents provided with each vector.
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Accessory Products
Accessory Products
The table below lists products that may be used with the Tag-On-Demand� System. Antibodies are available from Invitrogen to facilitate detection of the p64 (c-myc) protein expressed from the control plasmid. Depending on the vector used to express the gene of interest, reagents are available from Invitrogen to detect your C-terminally-tagged recombinant protein. For a list of Invitrogen expression vectors compatible with the Tag-On-Demand� System, see the Appendix, page 28. For more information about each vector, refer to our Web site (www.invitrogen.com) or call Technical Service (see page 31).
Item Amount Catalog no.
Lipofectamine� 2000 Reagent 0.75 ml 11668-027
1.5 ml 11668-019
Opti-MEM® I Reduced Serum Medium 100 ml 31985-062
500 ml 31985-070
Phosphate-Buffered Saline (PBS), pH 7.4 500 ml 10010-023
Anti-myc Antibody 50 µl* R950-25
Anti-myc-HRP Antibody 50 µl* R951-25
Anti-myc-AP Antibody 125 µl* R952-25
Anti-myc-FITC Antibody 50 µl* R953-25
GFP Antiserum 50 µl* R970-01
WesternBreeze® Chromogenic Anti-Mouse Kit
1 kit WB7103
WesternBreeze® Chemiluminescent Anti-Mouse Kit
1 kit WB7104
*The amount of antibody supplied is sufficient for 25 Western blots or immunostaining reactions, as appropriate.
Introduction The Tag-On-Demand� System uses adenoviral-based stop suppression
technology to allow expression of an untagged (i.e. native) or C-terminally-tagged recombinant protein of interest in mammalian cells from a single expression vector. You may use one of the Tag-On-Demand� Gateway® vectors to quickly and easily generate an expression construct. Alternatively, you may use one of the many compatible expression vectors available from Invitrogen.
Advantages of the Tag-On-Demand� System
Using the Tag-On-Demand� System to facilitate transient expression of a C-terminally-tagged recombinant protein of interest in mammalian cells provides the following advantages:
• Provides a means to easily detect the expression or localization of a recombinant protein(s) for which there is no specific antibody available. Once tagged recombinant protein expression is verified, native protein expression experiments may be performed with the same construct.
• Uses adenovirus as a delivery vehicle, enabling efficient delivery of the Tag-On-Demand� Suppressor to a large variety of mammalian cell types.
• Delivers a transient source of the Tag-On-Demand� Suppressor to cells to minimize toxicity.
• Adaptable for high-throughput applications including rapid screening of a large number of genes for expression in a particular cell type.
Purpose of this Manual
This manual provides an overview of the Tag-On-Demand� System and provides general guidelines and instructions to:
• Generate an expression construct containing your gene of interest in a configuration suitable for expression using the Tag-On-Demand� System. If you are using one of the Tag-On-Demand� Gateway® vectors (see the next page), refer to the Tag-On-Demand� Gateway® Vectors manual for specific instructions.
• Store and handle the Tag-On-Demand� Suppressor Supernatant.
• Transduce the Tag-On-Demand� Suppressor Supernatant into mammalian cells, followed by immediate transfection of the expression construct to assay for expression of C-terminally-tagged protein.
• Transduce the Tag-On-Demand� Suppressor Supernatant into a stable cell line containing the expression construct of interest, and assay for C-terminally-tagged protein.
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Overview, continued
Tag-On-Demand� Gateway® Vectors
The Tag-On-Demand� Gateway® Kits are supplied with a Tag-On-Demand� Gateway® vector (i.e. pcDNA�6.2/GFP-DEST or pcDNA�6.2/V5-DEST) to allow generation of an expression construct compatible for use in the Tag-On-Demand� System. For detailed instructions to generate the expression construct using pcDNA�6.2/GFP-DEST or pcDNA�6.2/V5-DEST, refer to the Tag-On-Demand� Gateway® Vectors manual. This manual is supplied with the Tag-On-Demand� Gateway® Kits, but is also available for downloading from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 31).
Note: Catalog nos. K400-01 and K405-01 do not include a Tag-On-Demand� Gateway® vector.
The Tag-On-Demand� System facilitates transient expression of C-terminally-tagged and native recombinant proteins in mammalian cells from a single expression construct. The System is based on stop suppression technology originally developed by RajBhandary and colleagues (Capone et al., 1985), and consists of two major components:
• A mammalian expression vector into which the gene of interest will be cloned. This vector (e.g. pcDNA�6.2/V5-DEST or pcDNA�6.2/GFP-DEST) must be in a configuration that is compatible with expression of C-terminally-tagged recombinant protein using the Tag-On-Demand� System (see Generating the Expression Construct, page 9 for more information).
• The Tag-On-Demand� Suppressor Supernatant, a replication-incompetent adenovirus containing the human tRNAser suppressor gene. This tRNA suppressor has been mutated to recognize the TAG (amber stop) codon and decode it as a serine. When added to mammalian cells, the Tag-On-Demand� Suppressor Supernatant is transduced and provides a transient source of the tRNAser suppressor. If the expression construct encoding a gene of interest with a TAG stop codon is present in mammalian cells, the stop codon will be translated as serine, allowing translation to continue through any downstream reading frame (i.e. C-terminal tag). This results in production of a C-terminally-tagged fusion protein. For details on how the Tag-On-Demand� Suppressor Supernatant was generated, see below.
How the Tag-On-Demand� Suppressor Supernatant Was Generated
The Tag-On-Demand� Suppressor Supernatant was generated as follows.
1. A vector containing the gene encoding the tRNAser gene with its native promoter and terminator was obtained from Dr. Uttam RajBhandary at the Massachusetts Institute of Technology. This tRNAser gene has been mutated such that the anticodon recognizes the TAG (amber) stop codon, and is referred to as the tRNAser suppressor gene (Capone et al., 1985).
2. The tRNAser suppressor gene was PCR amplified and cloned into the pENTR�1A vector available from Invitrogen (Catalog no. 11813-011). This construct was used in a multimerization procedure Buvoli et.al., 2000 to generate a Gateway® entry clone containing 8 tandem copies of the tRNAser suppressor gene.
3. The entry clone was recombined with Invitrogen�s pAd/PL-DEST� vector (Catalog no. V494-20) using the Gateway® LR recombination reaction to generate a recombinant adenoviral expression clone, which was then used in Invitrogen�s ViraPower� Adenoviral Expression System (Catalog no. K4940-00) to produce recombinant adenovirus.
4. The recombinant adenovirus was CsCl-purified and titered to generate the Tag-On-Demand� Suppressor Supernatant.
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The Tag-On-Demand� System, continued
Advantages of Using an Adenoviral Delivery System
Using a recombinant adenovirus to deliver the tRNAser suppressor to mammalian cells provides the following advantages:
• Adenovirus has a very broad tropism and can be used to deliver the tRNAser suppressor to a large variety of mammalian cell lines and cell types
• Adenovirus can transduce mammalian cells with extremely high efficiency, resulting in delivery of the tRNAser suppressor to nearly 100% of mammalian cells
• The recombinant adenovirus does not integrate into the host genome; therefore, expression of the tRNAser suppressor gene is transient and will only persist for as long as the viral genome is present (typically 7-8 days after transduction). Note: Once transduced into mammalian cells, the tRNAser suppressor is expressed within 24 hours.
• The recombinant adenovirus is stable, and can be stored at -80°C for up to 6 months without loss of activity (see page 8 for recommended storage and handling instructions).
For more information about how adenovirus works, see below.
How Adenovirus Works
When the Tag-On-Demand� Suppressor Supernatant is added to mammalian cells, the recombinant adenovirus enters target cells by binding to the Coxsackie/Adenovirus Receptor (CAR) (Bergelson et al., 1997) and internalizing via integrin-mediated endocytosis (Russell, 2000). Once internalized, the recombinant adenovirus is actively transported to the nucleus, and begins to express the tRNAser suppressor genes. Note that in order for the Tag-On-Demand� System to function correctly, the mammalian host cell line must contain CAR. Most mammalian cell types express CAR, but levels vary. Depending on the amount of the CAR expressed in your mammalian cell line, transduction efficiencies may vary.
Transduction Use of the Tag-On-Demand� Suppressor Supernatant involves transduction of
the recombinant adenovirus into the mammalian cell. In this System, no viral replication occurs and no infectious viral progeny are generated. This means that mammalian cells are transduced, not infected.
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The Tag-On-Demand� System is not intended for long-term expression studies of tagged recombinant proteins as delivery of the tRNAser suppressor to mammalian cells can result in stop suppression of all genes that contain a TAG stop codon. This means that, in addition to the gene of interest, up to one-third of the stop codons in cellular genes can be suppressed while the tRNAser suppressor is present in the cell, resulting in potential toxicity to the cell over time.
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Biosafety Features of the Tag-On-Demand� Suppressor Supernatant
Introduction The recombinant adenovirus in the Tag-On-Demand� Suppressor Supernatant
includes a number of safety features designed to enhance its biosafety. These safety features are discussed in this section.
Information for European Customers
The Tag-On-Demand� Suppressor Supernatant contains recombinant adenovirus that is genetically modified in the following ways:
• Deleted in the E1 region
• Deleted in the E3 region
• Carries 8 tandem copies of the human tRNAser gene in which the anticodon has been mutated to recognize the UAG codon
As a condition of sale, this product must be used in accordance with all applicable local legislation and guidelines including EC Directive 90/219/EEC on the contained use of genetically modified organisms.
Biosafety Features of the Tag-On-Demand� Suppressor Supernatant
The recombinant adenovirus in the Tag-On-Demand� Suppressor Supernatant includes the following safety features:
• The entire E1 and E3 regions are deleted in the adenovirus genome, resulting in production of an adenovirus that is replication-incompetent when transduced into mammalian cells that do not express the E1a or E1b proteins (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995).
• Adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
Note: Because the adenovirus is present transiently in mammalian cells, the production of C-terminally-tagged protein resulting from stop suppression is also transient. As levels of adenovirus decrease, levels of C-terminally-tagged protein produced decrease.
• The Tag-On-Demand� Suppressor Supernatant is screened for the presence of wild-type replication-competent adenovirus (RCA) contamination using a supernatant rescue assay (Dion et al., 1996) with a detection sensitivity of one wild-type RCA per 109 recombinant adenovirus. Product is shipped with no detectable wild-type RCA.
Biosafety Level 2
This product is of biological origin to be used solely in R&D laboratories under the supervision of a technically qualified individual and in accordance with the requirements set forth at 40 CFR 725.234-235. NIH Guidelines for Research Involving Recombinant DNA (rDNA) Molecules should be followed where applicable. Components of the product pose moderate potential infection hazard to personnel and the environment. It is recommended that this product be handled in accordance with Biosafety Level 2 precautions as detailed in Biosafety in Microbiological and Biomedical Laboratories, latest edition. The publication is available through the Centers for Disease Control. Follow your institution�s disposal and inactivation controls for biological materials.
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Experimental Outline
Introduction Two options exist to express C-terminally-tagged recombinant protein using the
Tag-On-Demand� System.
• Option 1: Add the Tag-On-Demand� Suppressor Supernatant to cells, followed immediately by transfection of the expression construct containing your gene of interest to facilitate transient expression of tagged recombinant protein.
• Option 2: If you have already generated a stable cell line expressing your gene of interest, then simply transduce cells with the Tag-On-Demand� Suppressor Supernatant to facilitate expression of tagged recombinant protein.
Flow Chart for Option 1
The diagram below describes the general steps required to express your C-terminally-tagged recombinant protein in the Tag-On-Demand� System using simultaneous transduction and transfection.
The diagram below describes the general steps required to express your native orC-terminally-tagged recombinant protein in the Tag-On-Demand� System from a stable cell line containing your expression construct.
Storing and Handling the Tag-On-Demand� Suppressor Supernatant
Introduction General guidelines are provided in this section to store and handle the Tag-On-
Demand� Suppressor Supernatant.
Storage Guidelines
Follow the guidelines below to store and use the Tag-On-Demand� Suppressor Supernatant.
• Store the Tag-On-Demand� Suppressor Supernatant at -80°C.
• The Tag-On-Demand� Suppressor Supernatant is stable for at least 6 months if stored at -80°C. Re-titering before use is not necessary. If the Tag-On-Demand� Suppressor Supernatant has been stored at -80°C for longer than 6 months, you may want to re-titer the adenovirus as viral titers may decrease with long-term storage.
• Do not freeze/thaw the Tag-On-Demand� Suppressor Supernatant more than 3 times as viral titers can decrease with more than 3 freeze/thaw cycles.
Handling the Tag-On-Demand� Suppressor Supernatant
When using the Tag-On-Demand� Suppressor Supernatant, remember that you will be handling recombinant adenovirus. Follow the recommended Federal and institutional guidelines for working with BL-2 organisms.
• Perform all manipulations within a certified biosafety cabinet.
• Treat media containing adenovirus with bleach.
• Treat used pipets, pipette tips, and other tissue culture supplies with bleach or dispose of as biohazardous waste.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling the Tag-On-Demand� Suppressor Supernatant and media containing virus.
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Generating the Expression Construct
Introduction Before you can use the Tag-On-Demand� System to facilitate expression of
C-terminally-tagged recombinant protein, you will need to clone your gene of interest into a suitable mammalian expression vector. You may use any vector of your choice for expression, but to be compatible with the Tag-On-Demand� System, the expression construct must possess a number of features. These features are described below.
Factors to Consider When Creating the Expression Construct
When creating your expression construct, you may clone your gene of interest into any mammalian expression vector of your choice. Note that your insert and the expression vector must possess the following features.
Insert Requirements
• Your gene of interest must contain an ATG initiation codon in the context of a Kozak consensus sequence for proper initiation of translation in mammalian cells (Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of a Kozak consensus sequence is (G/A)NNATGG, where the ATG initiation codon is underlined. Note that other sequences are possible, but the G or A at position-3 and G at position +4 are the most critical for function (shown in bold).
• To facilitate proper stop suppression using the Tag-On-Demand� Suppressor Supernatant, your gene of interest must contain a stop codon encoded by the nucleotides, TAG.
Important: Inserts containing TAA or TGA stop codons are not compatible with the Tag-On-Demand� System.
• When cloning your insert into the expression vector, the gene must be cloned in frame with the C-terminal tag.
Expression Vector Requirements
• The expression vector must contain a C-terminal tag that terminates in a stop codon encoded by the nucleotides, TAA or TGA.
Exception: You may use an expression vector that does not contain a C-terminal tag, but you will need to incorporate a C-terminal tag into your insert. The C-terminal tag must terminate in a stop codon encoded by the nucleotides, TAA or TGA.
The diagram below illustrates the features necessary in your expression construct for compatibility with the Tag-On-Demand� System.
For high-level expression of your recombinant protein of interest from a vector containing the CMV promoter, we recommend using the pcDNA�6.2/V5-DEST or pcDNA�6.2/GFP-DEST Gateway® destination vector (see table below).
Vector Feature
pcDNA�6.2/V5-DEST Allows expression of your protein of interest fused to a V5 epitope tag
pcDNA�6.2/GFP-DEST Allows expression of your protein of interest fused to the GFP reporter gene (Chalfie et al., 1994; Crameri et al., 1996)
Each vector is available in a kit with the Tag-On-Demand� Suppressor
Supernatant, and allows generation of expression clones using the Gateway® Technology for use in the Tag-On-Demand� System (Catalog nos. K410-01 and K420-01). For more information about each vector, Gateway® Technology, and how to generate an expression construct, see the Tag-On-Demand� Gateway® Vectors manual. The manual is supplied with Catalog nos. K410-01 and K420-01, but is also available for downloading from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 31).
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If you wish to express a human gene of interest, consider using an Ultimate� Human ORF (hORF) Clone available from Invitrogen. The Ultimate� hORF Clones are fully sequenced clones provided in a Gateway® entry vector that is ready-to-use in a recombination reaction with the pcDNA�6.2/V5-DEST or pcDNA�6.2/GFP-DEST vector. All Ultimate� hORF Clones are fully compatible for expression in the Tag-On-Demand� System. For more information about the clones available, see our Web site (www.invitrogen.com) or call Technical Service (see page 31).
Other Compatible Invitrogen Vectors
Invitrogen has an extensive selection of mammalian expression vectors that are compatible for use with the Tag-On-Demand� System. Expression vectors with different promoters, C-terminal tags, and functional elements are available. For a list of these vectors, refer to the Appendix, page 29. For more information about each vector, see our Web site (www.invitrogen.com) or contact Technical Service (see page 31).
Note: We are always adding new expression vectors to our collection. For information about the latest new vectors and their compatibility with the Tag-On-Demand� System, see our Web site or contact Technical Service.
General Guidelines to Use the Tag-On-Demand� System
Introduction Once you have generated the expression construct containing your gene of
interest and have obtained purified plasmid DNA, you are ready to perform transfection and use the Tag-On-Demand� Suppressor Supernatant to facilitate expression of C-terminally-tagged protein. General factors to consider when using the Tag-On-Demand� Suppressor Supernatant are discussed below. We highly recommend that you read this section before beginning.
Factors Affecting Suppression
A number of factors can influence the TAG suppression efficiency, and consequently, the amount of C-terminally-tagged protein expressed. These factors include:
• The characteristics of your mammalian host cell line
• The health of your cells and experimental cell culture conditions
• The transfection method used to introduce your expression construct into the host cell line
• The transduction procedure used
• The multiplicity of infection used to transduce the Tag-On-Demand� Suppressor Supernatant into mammalian cells
Each of these factors is discussed further in this section.
Selecting a Cell Line
If you are expressing native recombinant protein from your expression construct, you may use any mammalian cell line of choice. However, to express the highest levels of C-terminally-tagged recombinant protein using the Tag-On-Demand� Suppressor Supernatant, your mammalian cell line must possess the following characteristics:
• Must express the Coxsackie/Adenovirus Receptor (CAR) to facilitate internalization of the Tag-On-Demand� Suppressor adenovirus (Bergelson et al., 1997; Russell, 2000). Most mammalian cell types express CAR, but levels vary. Transduction efficiencies will vary depending on the amount of the CAR expressed in your mammalian cell line, affecting the suppression efficiency and the amount of C-terminally-tagged protein expressed.
• Does not express the adenovirus E1 proteins (see Important Note on the next page).
• Must be amenable to efficient transfection of your expression construct using the transfection reagent of choice (see Transfection Method, page 12 for more information).
Cell lines or cell types that have been used successfully at Invitrogen to express C-terminally-tagged recombinant protein using the Tag-On-Demand� System include COS-7, CHO-S, HeLa, HT1080, and BHK-21, primary rat hippocampal and cortical neurons.
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General Guidelines to Use the Tag-On-Demand� System, continued
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The Tag-On-Demand� Suppressor adenovirus is deleted in the E1 region, rendering it replication-incompetent in any mammalian cells that do not express the E1 proteins. Do not perform expression studies in 293 cells or in any cell line that expresses the adenovirus E1 gene (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995). Viral replication will occur in these cells, leading to rapid death of the target cell within 1-2 days after infection with the Tag-On-Demand� Suppressor Supernatant.
Recommended Cell Culture Conditions
The health of your mammalian cell stock can affect the outcome of your expression experiment. For optimal results, follow the recommendations below when culturing cells:
• Make sure that your cells are healthy (i.e. exhibit > 95% viability) at the time of plating. Using poor quality cell stock (e.g. cells consistently allowed to become overgrown or confluent before passaging, growth media allowed to become yellow before re-feeding) can negatively impact the suppression efficiency and the amount of C-terminally-tagged protein expressed.
• Use freshly prepared media during the course of the expression experiment.
Transfection Method
You may use any method of choice to transfect your expression construct into mammalian cells. Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989), and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). Consult published literature or the supplier of your cell line for the recommended method of transfection and transfection reagent to use.
For high-efficiency transfection of a large variety of mammalian cell lines including those listed on the previous page, we recommend using Invitrogen�s cationic lipid-based reagent, Lipofectamine� 2000 Reagent (see below).
Lipofectamine� 2000 Reagent
Lipofectamine� 2000 Reagent (Ciccarone et al., 1999) is a proprietary, cationic lipid-based formulation designed for transfection of nucleic acids into eukaryotic cells. Using Lipofectamine� 2000 Reagent to transfect mammalian cells offers the following advantages:
• Provides the highest transfection efficiency in a wide variety of mammalian cell lines
• DNA-Lipofectamine� 2000 Reagent complexes can be added directly to cells in culture medium in the presence of serum
• Removal of complexes or medium change or addition following transfection are not required, although complexes can be removed after 4-6 hours without loss of activity
Lipofectamine� 2000 Reagent is available separately from Invitrogen (see page vii for ordering information). For more information, see our Web site (www.invitrogen.com) or call Technical Service (see page 31).
General Guidelines to Use the Tag-On-Demand� System, continued
Deciding Which Transduction Option to Use
As discussed previously, two options exist for using the Tag-On-Demand� Suppressor Supernatant to facilitate expression of tagged recombinant protein. Choose the option that best suits your needs (see table below).
If you want to� Then�
• Quickly screen for expression or localization (if possible) of your recombinant protein
• Screen for expression of a large number of genes
Transduce the mammalian cell line of choice with the Tag-On-Demand� Suppressor Supernatant, followed immediately by transfection of the expression construct (see Note below)
Screen for expression of your recombinant protein from a stable cell line
Transduce the stable cell line of choice with the Tag-On-Demand� Suppressor Supernatant
Many studies have demonstrated that adenovirus transduction performed simultaneously with plasmid transfection can increase plasmid-derived gene expression as well as reduce toxicity to the cell (Cotten et al., 1992; Curiel et al., 1991; Guy et al., 1995; Honda et al., 1996; Merwin et al., 1995). We have found that this is the case when using the optimized Tag-On-Demand� transduction and transfection procedure on pages 17-18.
Multiplicity of Infection (MOI)
To obtain TAG suppression and maximal expression of C-terminally-tagged protein, transduce the Tag-On-Demand� Suppressor Supernatant into your mammalian cell line of choice using a suitable multiplicity of infection (MOI). MOI is defined as the number of virus particles per cell and generally correlates with expression. For most applications, we recommend transducing cells at an MOI of 50. Use this recommended MOI as a starting point for your expression experiments. Depending on the cell line used and the nature of the gene of interest, you may want to vary the MOI used to optimize expression of your C-terminally-tagged recombinant protein (see the next page).
For an equation to calculate the amount of Tag-On-Demand� Suppressor Supernatant to use to obtain a certain MOI, see page 15.
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For the cell lines tested (i.e. COS-7, CHO-S, HeLa, HT1080, BHK-21, and primary rat hippocampal and cortical neurons), we have found that transducing the Tag-On-Demand� Suppressor Supernatant at an MOI of 50 followed by immediate transfection of the expression construct generally results in 50-80% suppression. This means that 50-80% of the recombinant protein of interest is expressed as C-terminally-tagged protein when compared to total recombinant protein (untagged plus tagged). Note that 100% suppression cannot be achieved at any MOI. Some untagged recombinant protein will always be expressed. For an example, see page 24.
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General Guidelines to Use the Tag-On-Demand� System, continued
Varying the MOI The % suppression (i.e. suppression efficiency) achieved when mammalian cells
are transduced at a particular MOI (e.g. MOI = 50) can vary and is dependent on a number of factors including:
• The amount of CAR expressed in the mammalian cell
• The nature of the gene being expressed
• The health of the cells at the time of transduction
• Phenotypic changes to the cells resulting from TAG suppression (see below)
Depending on the suppression efficiency and consequently, the amount of C-terminally-tagged recombinant protein expressed, you may optimize the % suppression achieved by varying the MOI. It is important to note that while you may increase the % suppression achieved by increasing the MOI, doing so may increase the likelihood of phenotypic changes to the cells.
Phenotypic Effects Delivery of the Tag-On-Demand� Suppressor Supernatant to mammalian cells
may, in some cases, result in phenotypic effects on the host cell (e.g. toxicity) since 1/3 of the endogenous stop codons (i.e. all genes containing TAG stop codons) can be suppressed. This leads to potential addition of extra amino acids to the C-termini of cellular proteins other than the heterologous protein of interest. For the cell lines tested, minimal phenotypic effects have been observed using the recommended transduction and transfection protocols on pages 17-18.
Transient Nature of the Tag-On-Demand� Suppressor Supernatant
Remember that the Tag-On-Demand� Suppressor Supernatant delivers a transient source of the tRNAser suppressor to the target cell. Since the Tag-On-Demand� adenovirus is replication-incompetent, it does not stably integrate into the genome of the target cell, and will be diluted out gradually as cell division occurs. This results in an overall decrease in tRNAser suppressor expression over time.
When assaying for your C-terminally-tagged recombinant protein, we recommend performing detection 24 to 48 hours after transduction. During this time period, high levels of the tRNAser suppressor are expressed, thus allowing maximal expression of the C-terminally-tagged recombinant protein.
15
Using the Tag-On-Demand� Suppressor Supernatant
Introduction Guidelines and instructions are provided in this section to transduce your
mammalian cell line of choice with the Tag-On-Demand� Suppressor Supernatant to facilitate expression of your C-terminally-tagged recombinant protein.
Performing Simultaneous Transduction and Transfection
Once you have generated your expression construct, you can perform studies using the Tag-On-Demand� System to confirm expression of your recombinant protein or to determine where the protein is localized. You will transduce your mammalian cell line of choice with the Tag-On-Demand� Suppressor Supernatant, and follow immediately by transfecting your expression construct.
Transducing a Stable Cell Line
If you have already generated a stable cell line expressing your recombinant protein of interest, you may transduce cells with the Tag-On-Demand� Suppressor Supernatant to facilitate transient expression of tagged recombinant protein.
Recommended Guidelines
We generally transduce and transfect mammalian cells using the following conditions. Use these guidelines as a starting point for your experiments, and optimize conditions for your cell line and your protein of interest, as desired.
Condition Recommendation
Tissue culture plate size Use any size tissue culture plate of choice. We use 6-well, 24-well, or 96-well plates. For high-throughput applications, use 96-well plates.
Number of cells to transduce Plate cells such that they will be 90% confluent at the time of transduction.
Amount of Tag-On-Demand� Suppressor Supernatant to add
Sufficient to achieve an MOI of 50 (see below for an equation to calculate how much Supernatant to add).
Transfection reagent* Use Lipofectamine� 2000 Reagent (Catalog no. 11668-027).
Amount of plasmid DNA to transfect*
Amount recommended by the manufacturer of the transfection reagent you are using, and suitable for the scale of your transfection.
*If you are using another transfection reagent, follow the manufacturer�s recommendations for amount of DNA to transfect.
Calculating the Amount of Suppressor Supernatant to Add
Use the following formula to calculate how much Tag-On-Demand� Suppressor Supernatant to add to obtain a specific MOI. See the next page for an example.
MOI (pfu/cell) x number of cells Tag-On-Demand� Suppressor Supernant (ml) = viral titer (pfu/ml)
Note: The titer of the Tag-On-Demand� Suppressor Supernatant is 1 x 109 pfu/ml.
continued on next page
16
Using the Tag-On-Demand� Suppressor Supernatant, continued
Example We plate 8 x 104 COS-7 cells in a 24-well format and culture cells overnight at
37°C. On the following day, we wish to transduce cells with the Tag-On-Demand� Suppressor Supernatant using an MOI = 50. Assume that the number of cells has doubled during overnight culture so that the number of cells is now 2 x 8 x 104 = 1.6 x 105 cells. The titer of the Tag-On-Demand� Suppressor Supernatant is 1 x 109 pfu/ml.
50 pfu/cell x 1.6 x 105 cells Tag-On-Demand� Suppressor Supernant (ml) = 1 x 109 (pfu/ml)
= .008 ml = 8 µl to add to each well
Positive Control The pcDNA�6.2/GFP-GW/p64TAG plasmid is supplied with the Tag-On-
Demand� Suppressor Supernatant as a positive control for transduction, transfection, and expression. In this plasmid, the p64 protein (human c-myc) containing a TAG stop codon is cloned in frame with the cycle-3 GFP reporter gene (Chalfie et al., 1994; Crameri et al., 1996). Including the pcDNA�6.2/GFP-GW/p64TAG plasmid in your Tag-On-Demand� transduction and transfection experiment allows you to assay for cycle-3 GFP expression using fluorescence microscopy or c-myc expression using Western blot analysis (see page 21), and can help you evaluate your results. For a map and a description of the features of pcDNA�6.2/GFP-GW/p64TAG, see the Appendix, page 28.
Note: The Tag-On-Demand� V5 Gateway® Vector Kit (Catalog no. K420-01) does not include the pcDNA�6.2/GFP-GW/p64TAG control plasmid. If you have purchased this kit, use the pcDNA�6.2/V5-GW/p64TAG plasmid supplied with the kit as the positive control.
Materials Needed
You should have the following materials on hand before beginning:
• Your mammalian cell line of choice (make sure that cells are healthy and at >95% viability before use)
Reminder: Do not use 293 cells or any cell line that expresses E1
• Complete growth media for your cell line (use freshly-prepared media)
• Appropriately-sized tissue culture plates for your application (e.g. 24-well plate)
• Tag-On-Demand� Suppressor Supernatant (store at -80°C until use)
• Purified, plasmid DNA of the expression construct containing your gene of interest (if performing simultaneous transduction and transfection)
• pcDNA�6.2/GFP-GW/p64TAG or other positive control
• Lipofectamine� 2000 Reagent or other transfection reagent of choice (if performing transfection)
• Opti-MEM® I Reduced Serum Medium (if using Lipofectamine� 2000 Reagent; Catalog no. 31985-062)
continued on next page
17
Using the Tag-On-Demand� Suppressor Supernatant, continued
Transduction Procedure
Use this procedure to transduce your mammalian cell line with the Tag-On-Demand� Suppressor Supernatant. The procedure provides instructions to perform your experiment in a 24-well format. For other sized formats, see Scaling Transductions Up or Down, page 19 for recommended reagent volumes to use. We recommend including a positive control and a negative control (no virus) in your experiment to help you evaluate your results.
Example: If you are using COS-7 cells, see the table on page 19 for recommended seeding densities and reagent quantities.
1. One day before transduction (Day 1), plate cells in 500 µl of complete growth medium per well such that they will be 90% confluent at the time of transduction. Incubate cells at 37°C overnight.
2. On the day of transduction (Day 2), remove the growth medium from each well of cells and replace with 250 µl of fresh growth medium.
3. Thaw the Tag-On-Demand� Suppressor Supernatant and vortex briefly. Add the appropriate amount of adenovirus to each well. As a starting point, use an MOI of 50 (see page 15 for a formula to calculate how much Tag-On-Demand� Suppressor Supernatant to add). Return the cells to a 37°C incubator.
Reminder: When calculating how much Tag-On-Demand� Suppressor Supernatant to add, remember to take into account the doubling time of the cells when determining the total number of cells. For most dividing cell lines (e.g. COS-7, CHO), we assume that the number of cells present at the time of transduction has doubled from the amount plated.
4. If you are performing simultaneous transduction and transfection, proceed immediately to the Transfection Procedure, next page. If you are transducing a stable cell line, incubate cells for 5-6 hours at 37°C.
Note: Do not incubate cells for less than 5 hours as transduction efficiency will be decreased. Longer incubation time is possible (e.g. overnight), but will not increase the transduction efficiency and may increase cell toxicity.
5. After 5-6 hours, remove the medium containing virus from the cells and gently wash once with 500 µl of fresh, complete growth medium. Add 500 µl of fresh, complete growth medium to the cells and return them to a 37°C incubator.
6. Assay for C-terminally-tagged recombinant protein expression 24-48 hours post-transduction (Days 3-4), as desired.
continued on next page
18
Using the Tag-On-Demand� Suppressor Supernatant, continued
Transfection Procedure
Use this procedure to transfect your mammalian cells in a 24-well format with the expression construct of interest using Lipofectamine� 2000 Reagent. To determine the amount of DNA and Lipofectamine� 2000 Reagent to use for other sized formats, and for details and tips to optimize transfection, refer to the Lipofectamine� 2000 Reagent manual supplied with the product. If you are using another transfection reagent, transfect cells according to the manufacturer�s recommended procedure.
Example: If you are using COS-7 cells, see the table on the next page for recommended reagent quantities.
1. Once you have added the Tag-On-Demand� Suppressor Supernatant to mammalian cells, immediately prepare DNA-Lipofectamine� 2000 Reagent complexes as follows. For each transfection sample:
a. Dilute 500 ng of DNA in 50 µl of Opti-MEM® I Reduced Serum Medium without serum. Mix gently.
b. Mix Lipofectamine� 2000 Reagent gently before use, then dilute 1.5 µl in 50 µl of Opti-MEM® I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature.
c. After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine� 2000 Reagent (total volume is 100 µl). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-Lipofectamine� 2000 Reagent complexes to form. The solution may appear cloudy, but this will not inhibit the transfection.
2. Remove the cells from the incubator and add DNA-Lipofectamine� 2000 Reagent complexes directly to the growth medium containing Tag-On-Demand� Suppressor Supernatant.
3. Incubate cells at 37°C for 5-6 hours.
Note: Do not incubate for less than 5 hours as transfection efficiency will be decreased. Longer incubation time is possible (e.g. overnight), but will not increase the transfection efficiency and may increase cell toxicity.
4. After 5-6 hours, remove the medium containing virus from the cells and gently wash once with 500 µl of fresh, complete growth medium. Add 500 µl of fresh, complete growth medium to the cells and return them to a 37°C incubator.
5. Assay for C-terminally-tagged recombinant protein expression 24-48 hours post-transfection (Days 3-4), as desired.
Note: Increased toxicity may be observed if cells are incubated for longer than 48 hours post-transfection.
continued on next page
19
Using the Tag-On-Demand� Suppressor Supernatant, continued
Scaling Trans-ductions Up or Down
To transduce cells in different tissue culture formats, vary the amounts of cells and medium used in proportion to the difference in surface area (see table).
Culture Vessel
Surface Area per Well (cm2)
Relative Surface Area (vs. 96-well)
Volume of Plating Medium
Volume of Medium Containing Virus
96-well 0.3 0.2 100 µl 50 µl
24-well 2 1 500 µl 250 µl
6-well 10 5 2 ml 1 ml
Example: Recommended Conditions for COS-7 Cells
If you are performing your transduction and transfection experiments in COS-7 cells, see the table below for recommended seeding densities and reagent quantities for transduction and transfection in different tissue culture formats. Note that the suggested DNA quantities are for transfection using Lipofectamine� 2000 Reagent.
Condition 6-well 24-well 96-well
Seeding density 3 x 105 cells 8 x 104 cells 1 x 104 cells
MOI = 50 3 x 107 virus 8 x 106 virus 1 x 106 virus
Amount of plasmid DNA per well
2 µg 500 ng 320 ng
Amount of Lipofectamine� 2000 Reagent per well
6 µl 1.5 µl 1 µl
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Remember that the Tag-On-Demand� Suppressor Supernatant provides a transient source of the tRNAser suppressor, and that expression of recombinant protein from a transiently transfected plasmid generally peaks within 24-48 hours following transfection. To obtain maximal levels of C-terminally-tagged recombinant protein, we recommend harvesting your cells and assaying for recombinant protein expression between 24 and 48 hours post-transfection. Since expression conditions will vary depending on the nature of your protein of interest and its half-life, you will need to optimize conditions to obtain maximal levels of recombinant protein expression.
continued on next page
20
Using the Tag-On-Demand� Suppressor Supernatant, continued
What You Should See
If you assay for your recombinant protein using Western blot analysis or immunofluorescence, what you see will depend on which antibody you use to detect the recombinant protein.
• If you use an antibody to the recombinant protein, you should be able to detect both untagged and C-terminally-tagged recombinant protein, allowing determination of the suppression efficiency, if desired.
• If you use an antibody to the C-terminal tag, you will only be able to detect the C-terminally-tagged recombinant fusion protein.
Examples To demonstrate the use of the Tag-On-Demand� Suppressor Supernatant to
facilitate expression of C-terminally-tagged recombinant protein, see the experiments described in Examples of Expected Results, pages 23-25. Note that the data are presented for illustrative purposes only. Your results may vary depending on the nature of your protein and the host cell line.
Assaying for GPF-Tagged p64TAG Protein
If you have included the pcDNA�6.2/GFP-GW/p64TAG control plasmid in your transduction and transfection experiment, you may assay for GFP-tagged recombinant p64TAG expression in a number of ways. See Detecting GFP-Tagged p64TAG Protein, next page.
21
Detecting GFP-Tagged p64TAG Protein
Introduction This section provides a review of various methods available to detect the GFP-
tagged p64TAG protein expressed from the pcDNA�6.2/GFP-GW/p64TAG control plasmid.
Detecting GFP Expression in vivo
You may detect expression of the GFP-tagged p64TAG fusion protein in vivo using fluorescence microscopy. Since the GFP-tagged p64TAG protein is expressed from the strong CMV promoter, fusion protein is generally detectable within 24 hours after transfection.
To detect fluorescent cells, it is important to pick the best filter set to optimize detection (see below for recommended filter sets). The primary excitation peak of cycle-3 GFP is at 395 nm. There is a secondary excitation peak at 478 nm. Excitation at either of these wavelengths yields a fluorescent emission peak with a maximum at 507 nm (see figure below). Note that the quantum yield can vary as much as 5- to 10-fold depending on the wavelength of light that is used to excite the GFP fluorophore.
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Recommended Filter Sets for GFP Detection
Use of the best filter set will insure that the optimal regions of the cycle-3 GFP spectra are excited and passed (emitted). For best results, use a filter set designed to detect fluorescence from wild-type GFP (e.g. Omega Optical XF76 Filter; see www.omegafilters.com).
You may use FITC filter sets to detect cycle-3 GFP fluorescence. but note that these are not optimal and fluorescent signal may be weaker. For example, the FITC filter set that we use excites cycle-3 GFP with light from 460 to 490 nm, covering the secondary excitation peak. The filter set passes light from 515 to 550 nm, allowing detection of most but not all of the cycle-3 GFP fluorescence.
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Most tissue culture media fluoresce because of the presence of riboflavin (Zylka and Schnapp, 1996) and may interfere with detection of cycle-3 GFP fluorescence. To alleviate this problem, remove the growth medium and replace with Phosphate-Buffered Saline (PBS; Catalog no. 10010-023) before assaying for GFP fluorescence. If cells are being cultured further after assaying, remove the PBS and replace with fresh growth medium prior to re-incubation.
You can assay for GFP-tagged p64TAG recombinant protein expression by Western analysis using the following antibodies available from Invitrogen.
• To detect both untagged and GFP-tagged p64TAG protein, use one of the Anti-myc Antibodies.
• To detect GFP-tagged p64TAG protein only, use the GFP Antiserum.
For more information about the antibodies available, see our Web site (www.invitrogen.com) or call Technical Service (see page 31). For ordering information, see page vii.
Preparing Cell Lysates to Assay for p64 Protein
When preparing cell lysates to assay for native or GFP-tagged p64 protein, note that procedures using NP-40 lysis are not effective in releasing p64 protein. Since p64 is localized in the nucleoli, harsher lysis procedures using RIPA or SDS-PAGE sample buffer are required to adequately solubilize p64 in total cell lysates. When preparing cell lysates to assay for p64 protein, we recommend using the following procedure.
1. Wash cell monolayers once with Phosphate-Buffered Saline (PBS, Catalog no. 10010-023).
2. Add 1X SDS-PAGE Sample Buffer (see below for a recipe) to each well containing cells. For a 24-well plate, use 100 µl of 1X SDS-PAGE Sample Buffer per well.
3. Use a pipet tip to loosen lysed cells from plate and transfer to a 1.5 ml microcentrifuge tube. Lysates will be viscous.
4. Heat samples at 70°C for 10 minutes. Every few minutes, vortex the sample and briefly centrifuge.
5. Load 5 µl of cell lysate onto an SDS-PAGE gel and electrophorese.
Note: The GFP-tagged p64TAG protein has a molecular weight of approximately 77.2 kDa.
Introduction This section presents several sample experiments performed using the Tag-On-
Demand� System to facilitate expression of C-terminally-tagged recombinant proteins of interest.
Expected Results: Example 1
In this experiment, the following ORFs were amplified to contain a TAG stop codon, and cloned into the pENTR/D-TOPO® Gateway® vector to generate entry clones. The entry clones were then transferred into the pcDNA�6.2/GFP-DEST vector using the Gateway® LR recombination reaction to create expression clones.
ORF Protein Genbank Accession # Cellular Localization
1 Human CGI-130 BC003357 Cytoplasm
2 Human nuclear splicing factor
BC000997 Nucleus
3 Human c-myc BC000141 Nucleoli COS-7 cells were transduced with the Tag-On-Demand� Suppressor Supernatant
at an MOI of 50 followed by transfection with the pcDNA�6.2/GFP-DEST expression constructs using the procedure on pages 17-18. Twenty-four hours post-transfection, GFP fluorescence was assayed using fluorescence microscopy. Fluorescent micrographs for each expression construct are shown below.
ORF 1 ORF 2 ORF 3 Results: For all three proteins above, use of the Tag-On-Demand� System results
in expression of detectable levels of GFP-tagged recombinant protein as measured by GFP fluorescence by 24 hours post-transfection. Also, the GFP-tagged recombinant protein is correctly localized to the appropriate cellular organelle.
The expression construct containing ORF3 (BC000141) in Example 1, above is the same construct as the control pcDNA�6.2/GFP-GW/p64TAG plasmid included with Catalog nos. K400-01, K405-01, and K410-01.
continued on next page
24
Examples of Expected Results, continued
Expected Results: Example 2
In this experiment, COS-7 cells were transduced with the Tag-On-Demand� Suppressor Supernatant at various MOIs following the procedure on page 17, and simultaneously transfected with the pcDNA�6.2/GFP-GW/p64TAG plasmid using Lipofectamine� 2000 Reagent and the procedure on page 18. Twenty-four hours post-transfection, cell lysates were prepared and analyzed by Western blot using the Anti-myc Antibody and the WesternBreeze® Chemiluminescent Anti-Mouse Kit (Catalog no. WB7104) to detect native and GFP-tagged p64TAG (c-myc) protein.
1 2 3 4 5 6
Lane 1. MagicMark� MW Standard
Lane 2. Untransfected COS-7 cells
Lane 3. MOI = 0
Lane 4. MOI = 50
Lane 5. MOI = 100
Lane 6. MOI = 200
Results:
• GFP-tagged c-myc protein is produced and detectable by Western blot within 24 hours post-transfection. The % suppression achieved is > 80% when transducing cells at an MOI ≥ 50.
• In this experiment, increasing the MOI has little effect on the suppression efficiency. Maximal levels of GFP-tagged c-myc protein are produced using an MOI = 50.
c-myc-GFP
c-myc
25
Examples of Expected Results, continued
Expected Results: Example 3
In this experiment, 96 of Invitrogen�s Ultimate� Human ORF Clones encoding 96 different kinases were transferred into the pcDNA�6.2/V5-DEST vector using the Gateway® LR recombination reaction to generate expression clones. The expression constructs were purified, and the plasmid DNA (ranging from 20 ng to 300 ng) was transfected using Lipofectamine� 2000 Reagent into COS-7 cells (plated in 96-well format) that had been transduced with the Tag-On-Demand� Suppressor Supernatant at an MOI of 50 following the procedure on pages 17-18. Forty-eight hours post-transfection, cell lysates were prepared and analyzed by Western blot using the Anti-V5 Antibody (Invitrogen, Catalog no. R961-25) and the WesternBreeze® Chemiluminescent Anti-Mouse Kit (Catalog no. WB7104) to detect V5-tagged recombinant protein. Note that native recombinant protein is not detected.
The figure below shows the results from 6 out of 96 of the expression constructs. 1 2 3 4 5 6
Results:
• V5-tagged recombinant protein is produced and detectable by Western blot within 48 hours post-transfection.
• The levels of V5-tagged recombinant protein produced vary widely from gene to gene. This is expected since transfection and expression conditions are not optimized for each gene (see below) and can vary depending on the nature of the gene of interest.
In the experiment above, the amount of plasmid DNA transfected and the amount of cell lysate loaded on the polyacrylamide gel were not quantitated for each sample (i.e. transfection and expression conditions were not optimized). In addition, antibodies to each of the 96 different kinase proteins do not exist. This experiment demonstrates the utility of the Tag-On-Demand� System to quickly screen and analyze the expression of large numbers of recombinant proteins for which antibodies do not currently exist.
Ultimate� Human ORF Clones
For more information about the human genes available in our Ultimate� Human ORF Clone Collection, see our Web site (www.invitrogen.com) or call Technical Service (see page 31).
Introduction Use the information in this section to troubleshoot your expression experiments
using the Tag-On-Demand� Suppressor Supernatant.
Problem Reason Solution
Low suppression efficiency
• Cells not healthy when plated
• Media used was not fresh
• Cells transduced with too little virus (i.e. low MOI)
• Cell line expresses low levels of CAR
• Cells not transduced for a sufficient length of time
• Make sure that cells are healthy and > 95% viable before plating.
• Use fresh media during the experiment.
• Increase the MOI. We recommend transducing cells at an MOI of 50.
• Use a cell line that expresses suitable levels of CAR (e.g. COS-7, CHO, HeLa).
• Transduce cells with the Tag-On-Demand� Suppressor Supernatant for 5-6 hours.
Phenotypic effects observed
• Cells incubated for too long after transduction and transfection
• Cells sensitive to transduction and transfection procedure
• Assay for recombinant protein expression 24-48 hours after transduction and transfection.
• Perform expression experiments in a different mammalian cell line. Alternatively, you can make a stable cell line containing your expression construct, then perform the transduction.
Poor transfection efficiency • Use a transfection reagent that provides high efficiency transfection in your cell line (e.g. Lipofectamine� 2000 Reagent).
• Follow the manufacturer�s instructions for the transfection reagent you are using.
Low expression of C-terminally-tagged recombinant protein
Recombinant protein expression assayed too early or too late
Assay for recombinant protein expression between 24 and 48 hours post-transfection.
• Store the Tag-On-Demand� Suppressor Supernatant at -80°C.
• Do not freeze/thaw the Tag-On-Demand� Suppressor Supernatant more than 3 times.
Tag-On-Demand� Suppressor Supernatant frozen and thawed too many times
Do not freeze/thaw the Tag-On-Demand� Suppressor Supernatant more than 3 times.
continued on next page
27
Troubleshooting, continued
Transient Expression, continued
Problem Reason Solution
Insert contains a TAA or TGA stop codon
Generate a new expression construct, making sure that your native gene of interest terminates with a TAG stop codon.
Insert not cloned in frame with the C-terminal tag
Generate a new expression construct, making sure that your gene of interest containing the TAG stop codon is cloned in frame with the C-terminal tag.
Waited too long after trans-duction to assay for protein
Assay for C-terminally-tagged protein between 24 and 48 hours after transfection.
Tag-On-Demand� Suppressor Supernatant not added
Transduce cells with an appropriate amount of the Tag-On-Demand� Suppressor Supernatant (e.g. MOI = 50) prior to transfection of the expression construct.
No expression of C-terminally-tagged protein
Cell line does not express CAR Use a cell line that expresses CAR.
C-terminal fusion protein too large
A non-TAG stop codon was not placed downstream of the C-terminal tag
Generate a new expression construct, making sure that the C-terminal tag terminates with a TAA or TGA stop codon.
Cells not healthy Make sure that cells are healthy and >95% viable before beginning expression experiments.
Some cell toxicity observed
Too much Tag-On-Demand� Suppressor Supernatant used for transduction
Decrease the amount (i.e. MOI) of the Tag-On-Demand� Suppressor Supernatant used for transduction.
Cell lysis or death observed
Used the 293 cell line or another cell line that expresses E1 for experiments
Do not use the 293 cell line or any cell line that expresses the E1 proteins for expression. Viral replication will occur and cause cell death within 1-2 days after infection.
28
Appendix
Map of pcDNA�6.2/GFP-GW/p64TAG
Description pcDNA�6.2/GFP-GW/p64TAG is a 7103 bp control vector expressing the p64
(human c-myc) protein, and was generated using the Gateway® LR recombina-tion reaction between an entry clone containing the human c-myc gene (Bernard et al., 1983; Colby et al., 1983; Watt et al., 1983) and pcDNA�6.2/GFP-DEST. Note: The c-myc gene encodes a protein with an expected molecular weight of 48 kDa, however, the native protein actually runs at 64 kDa on an SDS-PAGE gel. The GFP-tagged p64TAG protein has a molecular weight of approximately 77.2 kDa.
Map of pcDNA�6.2/GFP-GW/p64TAG
The map below shows the elements of pcDNA�6.2/GFP-GW/p64TAG. The complete sequence of the vector is available from our Web site (www.invitrogen.com) or by calling Technical Service (see page 31).
Invitrogen Vectors Compatible with the Tag-On-Demand� System
Introduction A large number of Invitrogen�s mammalian expression vectors are compatible
for use with the Tag-On-Demand� System. These vectors contain:
• A C-terminal tag
• A non-TAG stop codon (i.e. TAA or TGA) downstream of the C-terminal tag.
To express a tagged recombinant protein from one of these vectors, remember that you must:
• Include a TAG stop codon in your gene of interest
• Clone the gene in frame with C-terminal tag
Invitrogen Vectors A list of Invitrogen expression vectors compatible for use with the Tag-On-
Demand� System is provided below. Vectors are available that facilitate cloning of your gene of interest using the Gateway® Technology, TOPO® Cloning, or restriction enzyme digestion and ligation. For more information on any of these vectors, see our Web site (www.invitrogen.com) or call Technical Service (see page 31).
Vector Catalog no.
For viral expression
pLenti4/V5-DEST� K4980-00
pLenti6/V5-DEST� K4950-00
pLenti6/UbC/V5-DEST� K4990-00
pLenti6/V5-D-TOPO® K4960-00
pAd/CMV/V5-DEST K4930-00
For expression from a specific genomic locus using the Flp-In� System
Invitrogen Vectors Compatible with the Tag-On-Demand� System, continued
Invitrogen Vectors, continued
Vector Catalog no.
For constitutive expression from the CMV promoter
pcDNA�6.2/V5-DEST K420-01*
pcDNA�3.2/V5-DEST 12489-019
pcDNA�-DEST40 12274-015
pcDNA6.2/V5-GW/D-TOPO® K2460-20
pcDNA3.2/V5-GW/D-TOPO® K2440-20
pcDNA3.1D/V5-His-TOPO® K4900-01
pcDNA�3.1/V5-His-TOPO® K4800-01
pcDNA�3.1/V5-His V810-20
pcDNA�3.1/myc-His V800-20
pcDNA�3.1(-)/myc-His V855-20
pcDNA�4/V5-His V861-20
pcDNA�4/myc-His V863-20
pcDNA�6/V5-His V220-20
pcDNA�6/myc-His V221-20
For constitutive expression from the EF-1α promoter
pEF6/V5-His-TOPO® K9610-20
pEF1/V5-His V920-20
pEF1/myc-His V921-20
pEF4/V5-His V941-20
pEF4/myc-His V942-20
pEF6/V5-His V961-20
pEF6/myc-His V962-20
For constitutive expression from the UbC promoter
pUB6/V5-His V250-20
For constitutive secreted expression
pSecTag2 V900-20
pSecTag2/Hygro V910-20
For fusion to the GFP reporter gene
pcDNA�6.2/GFP-DEST K410-01*
pcDNA�-DEST47 12281-010
pcDNA3.1/CT-GFP-TOPO® K4820-01
*Includes the Tag-On-Demand� Suppressor Supernatant
31
Technical Service
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Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation�s liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order.
Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Service Representatives.
Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.
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Purchaser Notification
Introduction Use of the Tag-On-Demand� Suppressor Supernatant is covered under a number of different licenses including those detailed below.
Limited Use Label License No. 19: Gateway® Cloning Products
The purchase of this product conveys to the buyer the non-transferable right to use thepurchased amount of the product and components of the product in research conductedby the buyer (whether the buyer is an academic or for profit entity). The purchase of thisproduct does not convey a license under any method claims in the foregoing patents orpatent applications, or to use this product with any recombination sites other than thosepurchased from Life Technologies Corporation or its authorized distributor. The right touse methods claimed in the foregoing patents or patent applications with this product forresearch purposes only can only be acquired by the use of ClonaseTM purchased fromLife Technologies Corporation or its authorized distributors. The buyer cannot modifythe recombination sequence(s) contained in this product for any purpose. The buyercannot sell or otherwise transfer (a) this product, (b) its components, or (c) materialsmade by the employment of this product or its components to a third party or otherwiseuse this product or its components or materials made by the employment of this productor its components for Commercial Purposes. The buyer may transfer information ormaterials made through the employment of this product to a scientific collaborator,provided that such transfer is not for any Commercial Purpose, and that such collab-orator agrees in writing (a) not to transfer such materials to any third party, and (b) touse such transferred materials and/or information solely for research and not forCommercial Purposes. Notwithstanding the preceding, any buyer who is employed in anacademic or government institution may transfer materials made with this product to athird party who has a license from Life Technologies under the patents identified aboveto distribute such materials. Transfer of such materials and/or information tocollaborators does not convey rights to practice any methods claimed in the foregoingpatents or patent applications. Commercial Purposes means any activity by a party forconsideration and may include, but is not limited to: (1) use of the product or itscomponents in manufacturing; (2) use of the product or its components to provide aservice, information, or data; (3) use of the product or its components for therapeutic,diagnostic or prophylactic purposes; or (4) resale of the product or its components,whether or not such product or its components are resold for use in research. LifeTechnologies Corporation will not assert a claim against the buyer of infringement of theabove patents based upon the manufacture, use or sale of a therapeutic, clinicaldiagnostic, vaccine or prophylactic product developed in research by the buyer in whichthis product or its components was employed, provided that none of (i) this product, (ii)any of its components, or (iii) a method claim of the foregoing patents, was used in themanufacture of such product. Life Technologies Corporation will not assert a claimagainst the buyer of infringement of the above patents based upon the use of thisproduct to manufacture a protein for sale, provided that no method claim in the abovepatents was used in the manufacture of such protein. If the purchaser is not willing toaccept the limitations of this limited use statement, Life Technologies is willing to acceptreturn of the product with a full refund. For information on purchasing a license to usethis product for purposes other than those permitted above, contact LicensingDepartment, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California92008. Phone (760) 603-7200.
Limited Use Label License No. 51: Blasticidin and the Blasticidin Selection Marker
Blasticidin and the blasticidin resistance gene (bsd) are the subject of U.S. Patent No.5,527,701 sold under patent license for research purposes only. For information on pur-chasing a license to this product for purposes other than research, contact LicensingDepartment, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California92008. Phone (760) 603-7200. Fax (760) 602-6500.
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Purchaser Notification
Limited Use Label License No. 55: Cycle 3 GFP
The ‘cycle3’mutant GFP was produced by Maxygen, Inc. using DNA shuffling tech-nology. Commercial licensing inquiries should be directed to: Affymax Research Institute,4001 Miranda Avenue, Palo Alto, CA 94304, U.S.A.
Limited Use Label License No. 127: GFP
This product and its use is the subject of one or more of U.S. Patent Nos. 5,491,084 and6,146,826, and foreign equivalents. This product is sold under license from ColumbiaUniversity. Rights to use this product are limited to research use only, and expresslyexclude the right to manufacture, use, sell or lease this product for use for measuring thelevel of toxicity for chemical agents and environmental samples in cells and transgenicanimals. No other rights are conveyed. Not for human use or use in diagnostic ortherapeutic procedures. Inquiry into the availability of a license to broader rights or theuse of this product for commercial purposes should be directed to Columbia InnovationEnterprise, Columbia University, Engineering Terrace-Suite 363, New York, New York10027.
Limited Use Label License No. 153: Adenoviral Technology for Expression of RNA Molecules
This product is sold under license from Transgene S.A., 11, rue de Molsheim, Strasbourg,France, under which Life Technologies has been granted a limited right to provideproducts for research purposes. Your use of this product constitutes your agreement touse this product for internal research purposes only and not for any clinical, therapeutic,prophylactic, diagnostic or production use. If you do not agree to be bound by these terms,return the unopened container(s) to Life Technologies for a full refund.
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Product Qualification
Tag-On-Demand� Suppressor Supernatant
The Tag-On-Demand� Suppressor Supernatant is qualified as follows:
• The titer is verified by plaque assay.
• The Tag-On-Demand� Suppressor Supernatant is screened for the presence of wild-type replication-competent adenovirus (RCA) contamination using a supernatant rescue assay (Dion et al., 1996) with a detection sensitivity of one wild-type RCA per 109 recombinant adenovirus. Product is shipped with no detectable wild-type RCA.
• The Tag-On-Demand� Suppressor Supernatant is functionally qualified as follows:
1. On day 1, COS-7 cells are plated in 24-well plates at a density of 8 x 104 per well.
2. On day 2, cells are transduced with the Tag-On-Demand� Suppressor Supernatant at an MOI of 50 and transfected with the control pcDNA�6.2/GFP-GW/p64TAG plasmid using Lipofectamine� 2000 Reagent following the procedures on pages 17-18.
3. Twenty-four hours after transfection, cells are assayed for:
a. GFP-tagged p64TAG expression in vivo using fluorescence microscopy
b. Native p64TAG and GFP-tagged p64TAG expression using Western analysis with the Anti-myc Antibody and the WesternBreeze® Chemiluminescent Anti-Mouse Kit. Using densitometry, the % suppression is quantitated by comparing the amount of GFP-tagged protein produced to the amount of total protein produced (untagged + GFP-tagged protein). At least 50% suppression must be obtained.
pcDNA�6.2/GFP-GW/p64TAG Control
The structure of the vector is verified by restriction enzyme analysis.
36
References
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